Direct molecular analysis of Malassezia species from the clinical samples of patients with pityriasis versicolor

Background and Purpose: Species identification of Malassezia using culture-dependent methods is time-consuming due to their fastidious growth requirements. This study aimed to evaluate a rapid and accurate molecular method in order to diagnose the pityriasis versicolor (PV) and identify Malassezia species from direct clinical samples. Materials and Methods: Skin scraping or tape samples from patients with PV and healthy volunteers as the control group were collected. Diagnosis of PV was confirmed by direct microscopic examination. The DNA extraction was performed according to the steel-bullet beating method. Polymerase chain reaction-restriction fragment length polymorphism assay using HhaI restriction enzyme was applied for the identification and differentiation of Malassezia species. Results: The PCR method was able to detect Malassezia in 92.1% of specimens which were also confirmed with microscopic examination. Statistically, a significant association was observed between the results of the two assays (P < 0.001). Moderate agreement was identified between the two methods to diagnose the PV in both populations (Kappa: 0.55). Considering microscopic examination as the gold standard method for confirmation of PV, the sensitivity, specificity, positive predictive value, and negative predictive value values of the PCR assay for recognition of PV were 85%, 75%, 92%, and 60%, respectively. M. globosa and M. restricta were the most prevalent species isolated from patients. Conclusion: In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to diagnose and identify Malassezia directly from clinical samples. Consequently, it can be said that the molecular-based method provides more facilities to identify fastidious species, such as M. restricta.


Introduction alassezia
species are lipid-dependent basidiomycetous yeasts with numerous functionally distinct strains that have recently been classified as Malasseziomycetes class.Among the different clinical manifestations of Malassezia species, Pityriasis versicolor (PV) is a wellknown superficial infection of the skin due to overgrowth or morphogenesis of yeasts to filamentous forms [1].
During the past years, molecular-based assays [2][3][4][5][6][7] for Malassezia detection/identification have revealed the ubiquitous presence of Malassezia on the skin in addition to pathogenic roles in different skin disorders [8].While these approaches have met with various degrees of success, most of them require cultivation to enhance sensitivity, which increases both the potential for cultural bias and the turnaround time for analysis.
In this regard, two culture-independent molecular methods for the detection and identification of Malassezia from clinical samples have been tested in recent years.However, these methods require either separate amplification with specific primer sets for each species [4,5,7] or sequencing which can be costly and time-consuming.
Therefore, the present study aimed to evaluate the rapid and accurate molecular method for the species identification of Malassezia directly from clinical samples by amplification of the D1/D2 domain using Polymerase chain reaction-restriction fragment length polymorphism (PCR−RFLP).

Materials and Methods
In this cross-sectional study, samples from the skin of 125 patients with PV who referred to the Medical Mycology Laboratory of Razi Hospital in Tehran were collected.Moreover, 17 healthy volunteer individuals were included as the negative control group.The diagnosis of PV was confirmed by microscopic observation of scales using potassium hydroxide (10% KOH) examination, which demonstrated the typical ovoid clusters of yeast cells and short filamentous.Following the confirmed diagnosis of PV, a part of the sample was stored at 25 °C for molecular analysis by PCR assay.
To identify the Malassezia species by molecular method, fungal genomic DNA was extracted manually from each clinical sample using steel bullet disruption [9].The D1/D2 domains of the 26S rRNA gene which produces approximately 580 bp fragments were amplified [6].The PCR reactions were performed in a 25 μL final volume containing 2×Taq Master Mix (final conc.1×) (Ampliqon RED, Denmark), 0.5 μM primer, and 4 μL (20 ng) of template DNA.Temperature conditions for PCR were 5 min at 95 °C, 37 cycles of 45 s at 95 °C, 45 s at 58 °C and 1 min at 72 °C, followed by 7 min at 72 °C.The PCR products were analyzed by electrophoresis on 1% (w/v) Tris-acetate-EDTA agarose gels containing 2.5-3% ethidium bromide, and visualized under a UV detector (UVITEC, UK).
The 142 PCR products of the D1/D2 domains were subjected to RFLP analysis with the HhaI enzyme (New England Biolabs, Beverly, MA, USA).Restriction reactions were carried out in 10 μL volumes containing 5 μL (200 ng) of PCR product, 2 μL of the restriction enzyme, 1μL of appropriate reaction buffer (final conc.1×), and 2 μL of distilled water at 37 °C for 24 h.Restriction patterns were compared with those of reference Malassezia strains and species-specific patterns established in the original publications [6].The DNA sequencing of D1/D2 domains was performed for 2 clinical samples that were confidently identified by PCR−RFLP, and for 8 clinical samples that could not be identified by PCR−RFLP.
The Chi-squared test was used to analyze the statistical difference between the ability of methods to detect Malassezia species in patients and healthy subjects.The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay were calculated.

Results
This study was performed on 142 participants from whom 104 (73.2 %) skin scales and 38 (26.8%) scotch tapes were collected.The PCR amplification of the partial D1/D2 region produced a single amplicon of the expected size (Figure 1A) for 102 samples.In comparison to a molecular analysis by PCR, more samples were positive by direct microscopic examination (102 vs. 110).The PCR method could detect Malassezia in 94 (92.1%) specimens which were also positive in microscopic examination.
Moreover, the PCR analysis detected Malassezia in eight samples that showed negative results using the direct microscopy method.Statistically, a significant association was observed between the results of the two assays (P˂0.001).Considering microscopic examination as the gold standard method for confirmation of PV, the diagnostic performance of the PCR assay for recognition of PV was calculated.The sensitivity, specificity, PPV, and NPV values of the PCR were 85%, 75%, 92%, and 60%, respectively.
Upon digestion of the amplified products with the HhaI restriction enzyme, two different restriction patterns could be distinguished.Both patterns matched exactly the HhaI restriction patterns predicted for Malassezia globosa (129 and 455 bp), and Malassezia restricta (580 bp).In line with these patterns, M. globosa and M. restricta were detected in 80 (85.1%) and 14 (14.9%)samples, respectively (Figure 1B).
Based on the sequencing data of the D1/D2 region for eight clinical samples that could not be distinguished by PCR−RFLP, M. globosa and M. restricta were identified with 100% identity in seven and one samples, respectively.Representative sequences of the DNA-sequenced Malassezia are  The results of species distribution in the study population showed that M. globosa was found in 67.7% (n=84) of the patients with PV, and 11.7% (n=2) of the healthy population.It should be noted that M. restricta was just identified in patients with PV (15; 12.1%).

Discussion
According to the results, molecular identification involving PCR−RFLP analysis had a remarkable ability for the direct identification of Malassezia in clinical samples.As can be seen in previous studies [10,11], the diagnosis of fungal infections using PCR-based methods, if performed directly on clinical samples, has a significant improvement, compared to the results obtained from the conventional methods.
The advantages of these methods include 1) the possibility of simultaneous detection and identification of the causative agent, 2) no need for culture in cases where the sample cannot be cultured for any reason, and 3) reducing the time required to obtain the result to one day.Prompt detection and identification of the causative agents of infection lead to the selection of the appropriate treatment by time, as well as the prevention of the induction of drug resistance.Therefore, in this study, it was attempted not only to diagnose PV infection directly from the clinical sample by PCR−RFLP method but also to identify the common Malassezia species that are the causative agents of this infection.
The PCR assay used in this study has high diagnostic characteristics, especially in terms of sensitivity and PPV that could be arising from the selection of suitable primers for PCR analysis.Regarding species identification of Malassezia, the general or species-specific primers are designed from different regions of the rRNA complexes so far [12,13] or the elongation factor 1 alpha genes [14].Consistent with previously reported results [15], the primers used in the current study amplified the D1/D2 region that was efficient for species identification of Malassezia yeasts.
Until now, many molecular methods have been introduced for the detection and identification of Malassezia species from clinical isolates.The most common methods include nested PCR [16], PCR−RFLP [17], and real-time PCR [3] with different levels of accuracy.Among the above-mentioned methods, the PCR−RFLP method is a sensitive, accurate, and fast (when using fast digest enzymes) method without requiring advanced tools that can detect microorganisms at the species and intra-species levels [18,19].
Among the other advantages of PCR−RFLP assay, it can be pointed out that there is no need for complete gene sequence information, no influence from the environmental agents, and high reproducibility [20].However, it should be noted that this method also has disadvantages, which include the high cost of some endonuclease enzymes and the reduction of sequencing accuracy if there is more than one mutation in the gene sequence detected by the enzyme.
It should be noted that some studies have used the D1/D2 domain for PCR−RFLP assays and identified Malassezia species by exposing the PCR product to two to three enzymes.It seems that the reason for the variety of species identified in the prior studies, compared to our study, is the use of more than one enzyme in the PCR−RFLP reaction.
Recent studies based on culture methods in different geographical regions of Iran have shown that M. globosa is the most prevalent species recovered from PV patients (frequency range: 36-43%) [24][25][26][27].In view of these obtained results, it should be noted that M. restricta species could not easily recover by conventional culture methods.However, the independent culture methods are capable of identification of M. restricta.Consequently, it is worth noting that molecular methods are more efficient to determine the species distribution of fungal agents in epidemiological studies [5,28].

Conclusion
In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to identify Malassezia directly from clinical samples.Moreover, the suggested molecular-based method provides more facilities to identify fastidious species, such as M. restricta.

Figure 1A .
Figure 1A.Agarose gel electrophoresis of D1/D2 region of PCR products.Before digestion with HhaI: lanes 1-7 are example samples.Lanes cr+ and cr-are positive and negative controls, respectively, and lane M is a 100-bp molecular size marker.Figure 1B.After digestion with HhaI: lanes 1 and 2 are M. globosa, lanes 3 and 4 are M. restricta, and lane M is a 100-bp molecular size marker.

Figure 1B .
Figure 1A.Agarose gel electrophoresis of D1/D2 region of PCR products.Before digestion with HhaI: lanes 1-7 are example samples.Lanes cr+ and cr-are positive and negative controls, respectively, and lane M is a 100-bp molecular size marker.Figure 1B.After digestion with HhaI: lanes 1 and 2 are M. globosa, lanes 3 and 4 are M. restricta, and lane M is a 100-bp molecular size marker.